| Hepatitis B virus (hepatitis B virus, HBV) infection is a common disease that is the hazard to human health seriously and has wide prevalence in the world. World Health Organization has reported that about2billion people around the world have been affected HBV, among whom350million people are with chronic HBV infection. About1million people died from hepatic failure, liver cirrhosis and primary hepatocellular carcinoma caused by HBV infection. Chronic hepatitis B is the risk factor to liver cirrhosis and liver cancer. The lack of effective means to clear the virus is a tough problem to chronic hepatitis B therapy. HBV infection pathogenic mechanism is complex and is that a series of host immune responses cause pathological immune damage in liver cell, which lead to various clinical manifestations. And the main immune effector cell is HBV antigen specific T cells, especially cytotoxic T lymphocytes (CTLs), which play a crucial role in viral clearance in liver cells. Patients with acute hepatitis B present a strong and polyclonal CTL response, leading to a quick elimination of infected virus. By contrast, the HBV-specific CTL response is weak and lack of polyclonal CTL responses, leading to incomplete elimination of infected virus. The molecular mechanism of the differences in HBV specific CTL response has not been well understood. CTL is CD8+T cells. Its specificity depends on T cell receptor (TCR). TCR αβ T cell is the main CD8T cell that participates in specific cellular immune response. Its diversity and specificity generate from the rearrangement of VDJ region of TCR α and β chain gene. Therefore, it is very important to understand HBV immune response mechanism thoroughly by revealing the composition characteristics of specific CTL TCR molecular.The aim of this study is to analyze TCR molecular characteristics of CTL by the acquisition of HBV antigen specific CTLs, and to turn non-specific T lymphocytes into HBV antigen specific CTLs which get antiviral activity through genetically modified expression of specific CTL TCR, which make for the deep clarification of HBV immune response mechanism and establish foundation for the further assessment of HBV specific CTL function and mechanism in the organism level (such as HBV/HLA-A2transgenic mice). Moreover, finally it provides important help for clinical development and improvement of immune therapy based in HBV antigen specific CTL to treat chronic hepatitis B.Objective:To clone the coding gene of HBV antigen specific CTL TCR and investigate the binding affinity of retrovirus-mediated expression of T lymphocyte receptors on HBV-specific cytotoxic T lymphocytes.Methods:1. Peripheral blood mononuclear cells were isolated from patients with HLA-A2restricted acute hepatitis B. Epitopic peptides (WLSLLVPFV, Env335-343) and cytokines were used for induction and enrichment of specific CTL expansion. Specific CTL were cloned after depuration by sorting of the pentamer-positive CD8+T lymphocytes by flow cytometry and proliferated by B cell trophczoite translated from radiated EBV with anti-CD3/CD28antibody.2. RNA was extracted from purified T cells. Specific TCR Vα and Vβ coding genes were then amplified by RT-PCR,5’RACE and3’RACE etc. after designing degenerate primers. The genes of α and β chain with dominant subtype were considered as target genes for research after analyzing many cloned sequences.3. TCR coding gene retrovirus vectors were constructed and connected with genes of α and β chain in the internal ribosome entry sites (IRES). Recombined pseudotype virus obtained from transfected packaging cells were assayed.4. HLA-A2+PBMCs of Jurkat T cell and healthy people proliferated by using IL-2and anti-CD3antibody were respectively transduced by recombined pseudotype virus. The expression level of specific TCR was assayed by flow cytometry after staining with pentamer, anti-CD3, anti-CD8antibody and anti-Vβ13TCR-PE.Results:1. Analysis of600more cloned TCR gene sequences showed that the proliferated HBV-specific CD8T cells from all6acute hepatitis B patients presented predominance in TCR usage of α and β chains, with2-4α chain families and1-4β chain families in each case. The α2, α14,α15, β3,β13and β23families were detected in one more cases. One case had β13chain for all tested clones. For the same α chain or β chain family, CDR3sequences tended to be identical in one case but to be different between cases.2. The total length gene sequence of two paired TCR Vα and Vβ chains, respectively named as α21β13and α15β13, were obtained from one of HLA-A2+patients with acute hepatitis B.3. Specific retrovirus SAMEN vector had been connected with TCR coding genes α21β13and α15β13after evaluation, which had constructed respectively recombine retrovirus vector SAMEN-α15β13and SAMEN-α21-β13.The titer of packaged recombinant retroviruses was1.5×105～5.0×105IU/ml.4. Fluorescence staining by anti-Vβ13TCR-PE targeting the specific TCR and HLA-A2restricted epitope-specific pentamer showed a positive expression of reconstructed TCR on T cell. The positive cells occupied1.06%～2.25%for Vβ13on Jurkat cells,1.03%～2.06%for Vβ13chain and1.05%～1.12%for the epitope-specific pentamer on T cells from healthy HLA-A2+subjects respectively. By contrast, only less than0.05%cells from healthy HLA-A2" subjects were positive for either Vβ13or the pentamer.Conclusion:1. HBV-specific CD8T cells with antigenic peptide-induced proliferation had predominance in the usage of TCR α and β chains. This property might be one of the important molecular factors influencing anti-HBV immunity.2. Correctly-paired CTL TCRα/β chain gene can be obtained from preponderant CTL through sorting of small amounts of specific CTLs by flow cytometry after the induction of epitope peptide. This may circumvent the difficulty in establishing long-term CTL clones.3. It is the first report to obtain HLA-A2restricted Env335-343epitope-specific CTL TCR encoding genes as we know. HBV-specific TCR can be expressed by gene transfer mediated by retrovirus, which endues non-specific T cell binding activity to Env335-343epitope. |
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